Solid-State NMR of a Large Membrane Protein by Paramagnetic Relaxation Enhancement.
ABSTRACT Membrane proteins play an important role in many biological functions. Solid-state NMR spectroscopy is uniquely suited for studying structure and dynamics of membrane proteins in a membranous environment. The major challenge to obtain high quality solid-state NMR spectra of membrane proteins is sensitivity, due to limited quantities of labeled high-molecular-weight proteins. Here we demonstrate the incorporation of paramagnetic metal (Cu(2+)) ions, through either EDTA or a chelator lipid, into membrane protein samples for rapid data collection under fast magic-angle spinning (MAS) and low power (1)H decoupling. Spectral sensitivity of DsbB (20 kDa), an integral membrane protein, more than doubles in the same experimental time due to (1)H T(1) relaxation enhancement by Cu(2+) ions, with DsbB native fold and active site intact. This technique can be implemented to acquire multidimensional solid-state NMR spectra for chemical shift assignments and structure elucidation of large membrane proteins with small sample quantities.
Article: Insight into the mechanism of the influenza A proton channel from a structure in a lipid bilayer.[show abstract] [hide abstract]
ABSTRACT: The M2 protein from the influenza A virus, an acid-activated proton-selective channel, has been the subject of numerous conductance, structural, and computational studies. However, little is known at the atomic level about the heart of the functional mechanism for this tetrameric protein, a His(37)-Trp(41) cluster. We report the structure of the M2 conductance domain (residues 22 to 62) in a lipid bilayer, which displays the defining features of the native protein that have not been attainable from structures solubilized by detergents. We propose that the tetrameric His(37)-Trp(41) cluster guides protons through the channel by forming and breaking hydrogen bonds between adjacent pairs of histidines and through specific interactions of the histidines with the tryptophan gate. This mechanism explains the main observations on M2 proton conductance.Science 10/2010; 330(6003):509-12. · 31.20 Impact Factor